Flow cytometry utilizes an optical technique that analyzes particles in a fluid mixture based on the particles' optical characteristics using a flow cytometer. Background information on flow cytometry is, for example, found in Shapiro, Practical Flow Cytometry, Third Ed. (Alan R. Liss, Inc. 1995), incorporated herein by reference. Conventional flow cytometers have been commercially available since the early 1970s and presently cost, for example, more than $120,000. They can be behemoths in size, occupying upwards of 13 cubic feet and weighing well over 200 pounds.
In conventional flow cytometers, as shown in FIGS. 1 and 2, sample fluid containing sample cells or microspheres having reactants on their surfaces is introduced from a sample tube into the center of a stream of sheath fluid. The sample fluid stream is injected into, at, or near, the center of the flow cell or cuvette. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the center of the measuring point. Typically, the cells or microspheres are in suspension in the flow cell.
A continuous wave laser 1900 focuses a laser beam on them as they pass through the laser beam by a flow of a stream of the suspension. Lasers in conventional flow cytometers often require shaping a round beam into an elliptical beam to be focused on the flow cell. As shown in FIG. 2, this elliptical beam is often formed from the round beam using a beam shaping prismatic expander 1960 located between the laser and the flow cell. When an object of interest 1905 in the flow stream is struck by the laser beam, certain signals are picked up by detectors. These signals include forward light scatter intensity and side light scatter intensity. In the flow cytometers, as shown in FIGS. 1 and 2, light scatter detectors 1930, 1932 are located opposite the laser (relative to the cell) to measure forward light scatter intensity, and to one side of the laser, aligned with the fluid-flow/laser beam intersection to measure side scatter light intensity.
In front of the forward light scatter detector 1930 can be an opaque bar 1920, called a beam stop, that blocks incident light from the laser. Thus, the beam stop ensures that as little of the beam as possible will interfere with the measurement by the forward light scatter detector of the relatively small amount of light which has been scattered, by the flow cell, at small angles to the beam. Forward light scatter intensity provides information concerning the size of individual cells, whereas side light scatter intensity provides information regarding the relative size and refractive property of individual cells.
Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 to HANSEN et al., incorporated herein by reference, have been used, for example, to automatically identify subclasses of blood cells. The identification was based on antigenic determinants on the cell surface which react to antibodies which fluoresce. The sample is illuminated by a focused coherent light and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify the cells.
As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al., incorporated herein by reference, some flow cytometers use fluorescent microspheres, which are beads impregnated with a fluorescent dye. Surfaces of the microspheres are coated with a tag that is attracted to a receptor on a cell, an antigen, an antibody, or the like in the sample fluid. So, the microspheres, having fluorescent dyes, bind specifically to cellular constituents. Often two or more dyes are used simultaneously, each dye being responsible for detecting a specific condition.
Typically, the dye is excited by the laser beam from a continuous wave laser 1900, and then emits light at a longer wavelength. As shown in FIG. 1, dichroic filters 1940 split this emitted light and direct it through optical detectors 1950, 1952, 1954 that can be arranged relative to the laser. The optical detectors 1950, 1952, 1954 measure the intensity of the wavelength passed through a respective filter. The fluorescence intensity is a function of the cells' absorption of fluorescent dye.
FIG. 2 depicts a prior art flow cytometer which uses beam splitters 1942, 1944, 1946 to direct light from the flow cell 1910 to photo-multiplier and filter sets 1956, 1958, 1959 and to side light scatter detector 1932. This flow cytometer employs a mirror 1970 to reflect forward light scatter to forward light scatter detector 1930.
However, I have determined that the properties of the fluorescent dyes themselves limit this flow cytometric technique to about three different wavelengths. The difference in energy, and hence wavelength, between an excitation photon and emission photon is known as Stokes shift. Generally, the larger the Stokes shift from the excitation wavelength, the broader and weaker the emission spectra.
At any given excitation wavelength, I have determined that there are often only a limited number of dyes that emit a spectrum of wavelengths narrow enough and sufficiently separated enough that they are individually measurable simultaneously. Of these, there are fewer dyes still that exhibit good quantum efficiency, for example, between 5 and 40%. Other values for quantum efficiency are also acceptable. For example, values of 75 to 80% are acceptable. Consequently, researchers in flow cytometry and other fields have been limited to roughly three fluorescent labels, namely, for green, yellow-orange, and red light.
The limitation on the number of fluorescent labels necessarily crimps the amount of analysis that can be done on any one sample. Therefore, for meaningful analysis, a larger quantity of sample is required and more runs of the sample through the flow cytometer must be performed. This necessarily increases the time needed to analyze the sample. However, time is often not available in an emergency room environment, for example, where a small blood sample must be screened simultaneously for many diagnostic indicators, including therapeutic and abused drugs, hormones, markers of heart attack and inflammation, and markers of hepatic and renal function. In addition, for efficiency reasons, it is desirable to minimize the testing time to increase the number of tests that can be performed over a predetermined time interval.
One way to overcome the limitation on the number of fluorescent labels, I have determined, is to use two lasers of different frequencies, each focused on a different spot along the flow stream. Such a configuration is called a multi-station flow cytometer. As a particle passes a first laser, up to three fluorescence measurements are taken. Then, as the particle passes the second laser, up to three more measurements are taken using a time-gated amplifier at a predetermined time interval after signals have been detected at the upstream observation point. FIG. 3 illustrates this method.
It should be noted that the upper pair of particles A, B show the lower pair of particles A, B at a later time as the particles progress upward through the flow cell; the particles themselves are the same. In this case, laser #1 strikes particle A. A detector for Laser #2 must wait for a particle to pass through the beam of Laser #2.
Despite this dual laser approach, I have determined that it is often impossible to know for certain whether the measurements are made on the same particle. Because the measurement events at the sets of detectors are separated temporally and spatially, I have discovered that, besides laser emission timing problems, even the slightest flow turbulence can mix particles in suspension, thereby increasing the likelihood that subsequent measurements are not made on the same particle as the previous measurements.
Further, particles in the sample fluid exhibit different velocities as they pass through the flow cell depending on their respective distances from the center of the sample fluid flow stream. Plainly, a an particle closer to the center would travel faster than a particle further away from the center. As such, it is difficult or impossible to be sure exactly when a particle detected by a detector for Laser #1 will pass through a beam of Laser #2.
Referring to FIG. 3, flow turbulence, for example, causes particle B to change places with particle A such that laser #2 strikes particle B, instead of particle A. By extension, this unacceptable problem compounds as lasers and detectors are added to the device.
Despite this flaw, such multiple illumination beam capabilities have been limited to expensive, complex sorters and are not typically found in smaller, less expensive instruments. Besides being large and expensive, such machines are often fully burdened in the clinical setting with CD4-CD8 lymphocyte analysis.
Compounding the above-mentioned shortcomings of existing devices and methods, I have discovered that existing methods of data collection and analysis thereof is tedious, slow, and non-real-time. That is, substantially simultaneous detection of multiple analytes, or of separately identifiable characteristics of one or more analytes, through single-step assay processes is presently not possible, or to the extent possible, has provided limited capability and thus has yielded unsatisfactory results. Reasons for these disappointing results include the following. First, the length of time typically required to enable detection and classification of multiple analytes is unacceptably long. Second, the prior art assays exhibited low analyte sensitivities, which often lead to significant analytical errors and unwieldy collection, classification, and analysis of prior art algorithms relative to large amounts of collected data.
An existing bead set separation method involves the following steps. First, a test tube having sample fluid and sets of reporter beads must be loaded into the flow cytometer and depress the “Acquire” button. Second, when the desired number of data events have been collected, the “Stop” button must be pressed. Third, a file containing the collected data must be saved to a hard drive of a computer. Fourth, a control and analysis software package must be opened. Fifth, the file must be loaded into the control and analysis software package. Sixth, an x-y plot of FL2 v. FL3 must be charted, where FL2 and FL3 are orange and red fluorescence classification parameters for the sets of beads. Seventh, the sets of beads in the plot, represented by clouds of dots, must be visually located and a polygon gate must be drawn around the first set of interest to eliminate stray data points. Eighth, the file must be filtered for events that fall within the polygon gate. Ninth, the statistics must be displayed and the mean value of FL1 must be noted, wherein FL1 is the green fluorescence measurement for the analyte of interest.
Tenth, FL1 to FL2 percent spill-over must be calculated and subtracted from the mean value of FL2 to correct the value of FL2. Eleventh, the corrected value of FL2 is used to look up manually which bead set was located in the polygon gate. Twelfth, the FL2 to FL1 percent spill-over is calculated and subtracted from the mean value of FL1. Thirteenth, the assay result is determined from the adjusted value of FL1. The previous thirteen steps are manually repeated for each remaining set of beads.
In addition to the tedium associated with the above-described bead set separation method, I have discovered that the subjectivity associated with estimating the boundaries of the polygon gates is unacceptable. The value of any assay using this method depends largely on the variable judgment of a lab technician. It is often impossible to separate some sets of beads because of overlap of bead regions on the FL2-FL3 plot. Moreover, because of FL1 to FL2 spill-over, the FL2 value of a subset increases sufficiently to overlap with other fluorescence values of other bead sets. Consequently, because of the spill-over, two subsets occupy substantially the same region, making them impossible to distinguish visually there between. The net result of these difficulties is the inability to determine during a sample run, the existence and quantity of an analyte of interest.
In view of the above, I have determined that it would be desirable to have a system and/or method for detecting multiple analytes in a fluid sample by flow cytometric analysis and for analyzing and presenting the data in real-time.
I have determined that it would be desirable to have such a system and/or method, which eliminates the variability of human judgment and subjectivity from the data collection and analysis by performing data collection, bead set classification, and analysis techniques all carried out substantially simultaneously or contemporaneously.
I have also determined that it would be desirable to have such a system and/or method using a flow analyzer that is a fraction of the size, weight, and cost of conventional flow cytometers. That is, I have determined that the current “mainframe-style” flow cytometer must be replaced by a “desktop-style” personal cytometer.
I have further determined that it would be desirable to have such a system that is many times as fast as conventional flow cytometers and yet requires a fraction of the sample volume demanded by the conventional flow cytometers.
I have also recognized a deficiency in the current approach to signal processing in flow cytometry, which uses peak detectors to measure an event. When a peak is found, the peak detectors are disabled while the peaks are measured and processed. “Dead time,” the time period during which events can pass through the laser focal point undetected, is highly problematic when the flow cytometer is being used to search for rare events.
Prior art methods, such as U.S. Pat. No. 5,550,058 to Corio et al., incorporated herein by reference, are largely unsuccessful. However, no known prior art method and/or system, including that of Corio et al., has reduced dead time to zero. For example, Corio et al. pre-qualifies an event electronically to reduce the chance that a rare event slips by during dead time. The Corio et al. system sorts particles at a selected yield/purity ratio which ratio can include an intermediate value of the maximum yield and the maximum purity.
Prior art systems and/or methods, which do not use peak detection, use an integrator to measure the area under the pulse. Again, events pass through the laser beam undetected while the measurement is made. Thus, use of an integrator also fails to reduce dead time to zero.
In view of the above-described dead time problem, I have determined that it would be desirable to have a system and/or method for detecting multiple analytes in a fluid sample that reduces dead time in flow analysis to zero.